Regulation of intracellular glucocorticoid concentrations

ABSTRACT

This invention relates to the interconversion of inactive 11-keto steroids with their active 11B-hydroxy equivalents, to methods by which the conversion of the inactive to the active form may be controlled, and to useful therapeutic effects which may be obtained as a result of such control. More specifically, but not exclusively, the invention is concerned with interconversion between cortisone and cortisol in humans.

[0001] This invention relates to the interconversion of inactive 11-ketosteroids with their active 11β-hydroxy equivalents, to methods by whichthe conversion of the inactive to the active form may be controlled, andto useful therapeutic effects which may be obtained as a result of suchcontrol. More specifically, but not exclusively, the invention isconcerned with interconversion between cortisone and cortisol in humans.

[0002] Glucocorticoids such as cortisol have a number of diverse effectson different body tissues. For example, the use of cortisol as ananti-inflammatory agent was described in our International PatentApplication WO 90/04399, which was concerned with the problem thattherapeutically administered cortisol tends to be converted in the bodyto inactive cortisone by 11β-hydroxysteroid denydrogenase enzymes. Ourearlier invention provided for the potentiation of cortisol by theadministration of an inhibitor of the 11β-dehydrogenase activity ofthese enzymes.

[0003] Another major physiological effect of cortisol is its antagonismto insulin, and it is known for example that high concentrations ofcortisol in the liver substantially reduce insulin sensitivity in thatorgan, thus tending to increase gluconeogenesis and consequently raisingblood sugar levels [1]. This effect is particularly disadvantageous inpatients suffering from impaired glucose tolerance or diabetes mellitus,in whom the action of cortisol can serve to exacerbate insulinresistance. Indeed, in Cushing's syndrome, which is caused by excessivecirculating concentrations of cortisol, the antagonism of insulin canprovoke diabetes mellitus in susceptible individuals [2].

[0004] As mentioned above, it is known that cortisol can be converted inthe body to cortisone by the 11β-dehydrogenase activity of11β-hydroxysteroid dehydrogenase enzymes. It is also known that thereverse reaction, converting inactive cortisone to active cortisol, isaccomplished in certain organs by 11β-reductase activity of theseenzymes. This activity is also known as corticosteroid 11β-reductase,cortisone 11β-reductase, or corticosteroid 11β-oxidoreductase.

[0005] It has only recently become apparent that there are at least twodistinct isozymes of 11β-hydroxysteroid dehydrogenase (collectivelyabbreviated as 11β-HSD, which term is used, where appropriate, in thisspecification). Aldosterone target organs and placenta express a highaffinity NAD⁻-dependent enzyme (11β-HSD2)[3]. This has beencharacterized in placenta and kidney [4, 5]and cDNA clones have beenisolated [6, 9]. 11β-HSD2 catalyses 11β-dehydrogenase activityexclusively [4, 7]. In contrast, the previously purified, liver derivedisozyme (11β-HSD1) is a lower affinity. NADP⁻/NADPH-dependent enzyme[10, 11]. Expression of 11β-HSD1 in a range of cell lines encodes eithera bi-directional enzyme [11, 12] or a predominant 11β-reductase [13, 15]which, far from inactivating glucocorticoids, regenerates active11β-hydroxysteroid from otherwise inert 11-keto steroid. 11β-reductaseactivity, best observed in intact cells, activates 11-keto steroid toalter target gene transcription and differentiated cell function [13,14]. 11β-HSD1 and 11β-HSD2 are the products of different genes and shareonly 20% amino acid homology [6, 7].

[0006] As far as the applicants are aware no previous attempts have beenmade to modify the action of 11β-reductase. We have now found that it ispossible to inhibit this activity in vivo, and in doing so we havecreated the possibility of a novel medicament for use in treating manyof the deleterious effects of glucocorticoid excess. In one aspect,therefore, the invention provides the use of an inhibitor of11β-reductase in the manufacture of a medicament for control of 11-ketosteroid conversion to 11β-hydroxysteroid in vivo.

[0007] As mentioned above, one of the major physiological effects ofcortisol is insulin antagonism in the liver, and in a specific aspectthe invention therefore provides the use of an inhibitor of11β-reductase in the manufacture of a medicament for inhibiting hepaticgluconeogenesis [1]. Cortisol promotes hepatic gluconeogenesis byseveral mechanisms, including antagonism of the effects of insulin onglucose transport, and interactions with insulin and glucose in theregulation of several enzymes which control glycolysis andgluconeogenesis. These include glucokinase. 6-phosphofructokinase,pyruvate kinase, phosphoenolpyruvate carboxykinase (PEPCK), andglucose-6-phosphatase. Inhibiting production of cortisol from cortisonein the liver therefore enhances hepatic glucose uptake and inhibitshepatic glucose production by several mechanisms [16]. Moreover, theinfluence of inhibiting 11β-reductase activity in the liver of patientswith insulin resistance or glucose intolerance may be greater than inhealthy subjects because in insulin resistance or deficiency theinfluence of cortisol on PEPCK has been shown to be great [17]; obesepatients secrete more cortisol [18]; insulin resistant patients are moresensitive to glucocorticoids [19]; and insulin down-regulates 11β-HSD1expression [15, 20] so that 11β-reductase activity may be enhanced inconditions of insulin resistance or deficiency.

[0008] Our studies have also shown that 11β-HSD1 is expressed in ratadipose tissue and in adipocyte cell lines in culture, where it converts11-dehydrocorticosterone to corticosterone (the rat equivalents of humancortisone and cortisol, respectively). This suggests that similar11β-reductase activity will be observed in human adipose tissue, withthe result that inhibition of the enzyme will result in alleviation ofthe effects of insulin resistance in adipose tissue in humans. Thiswould lead to greater tissue utilisation of glucose and fatty acids,thus reducing circulating levels. The invention therefore provides, in afurther aspect, the use of an inhibitor of 11β-reductase in themanufacture of a medicament for increasing insulin sensitivity inadipose tissue.

[0009] The results of our studies have encouraged us to believe thatinhibition of intracellular cortisol production will also lead toincreased insulin sensitivity in other tissues which are acted upon byinsulin, for instance skeletal muscle [21]. Inhibiting the 11β-reductasetherefore promises to reverse the effects of insulin resistance inmuscle tissue, and to promote the up-take of essential molecules such asglucose and free fatty acids into muscle cells with consequent improvedmuscle metabolism and reduction of circulating levels of glucose andfatty acids. In a further aspect, the invention therefore provides theuse of an inhibitor of 11β-reductase in the manufacture of a medicamentfor increasing insulin sensitivity in skeletal muscle tissue.

[0010] It is also known that glucocorticoid excess potentiates theaction of certain neurotoxins, which leads to neuronal dysfunction andloss. We have studied the interconversion between11-dehydrocorticosterone and corticosterone in rat hippocampal cultures,and have found (surprisingly in view of the damaging effects ofglucocorticoids) that 11β-reductase activity dominates over11β-dehydrogenase activity in intact hippocampal cells [22]. The reasonfor this activity is unknown, but this result indicates thatglucocorticoid excess may be controlled in hippocampal cells (and byextension in the nervous system in general) by use of an 11β-reductaseinhibitor, and the invention therefore provides in an alternative aspectthe use of an inhibitor of 11β-reductase in the manufacture of amedicament for the prevention or reduction of neuronal dysfunction andloss due to glucocorticoid potentiated neurotoxicity. It is alsopossible that glucocorticoids are involved in the cognitive impairmentof ageing with or without neuronal loss and also in dendriticattenuation [23-25]. Furthermore, glucocorticoids have been implicatedin the neuronal dysfunction of major depression. Thus, an inhibitor of11β-reductase could also be of value in these conditions.

[0011] It will be appreciated from the foregoing that the potentialbeneficial effects of inhibitors of 11β-reductase are many and diverse,and it is envisaged that in many cases a combined activity will bedemonstrated, tending to relieve the effects of endogenousglucocorticoids in diabetes mellitus, obesity (including centripetalobesity), neuronal loss and the cognitive impairment of old age. Thus,in a further aspect, the invention provides the use of an inhibitor of11β-reductase in the manufacture of a medicament for producing multipletherapeutic effects in a patient to whom the medicament is administered,said therapeutic effects including an inhibition of hepaticgluconeogenesis, an increase in insulin sensitivity in adipose tissueand muscle, and the prevention of or reduction in neuronalloss/cognitive impairment due to glucocorticoid-potentiatedneurotoxicity or neural dysfunction or damage.

[0012] From an alternative point of view, the invention provides amethod of treatment of a human or animal patient suffering from acondition selected from the group consisting of hepatic insulinresistance, adipose tissue insulin resistance, muscle insulinresistance, neuronal loss or dysfunction due to glucocorticoidpotentiated neurotoxicity, and any combination of the aforementionedconditions, the method comprising the step of administering to saidpatient a medicament comprising a pharmaceutically active amount of aninhibitor of 11β-reductase.

[0013] As mentioned previously, the factors which control the relativeactivities of 11β-dehydrogenase and 11β-reductase in differentconditions, especially by the 11β-HSD1 isozyme are poorly understood. Itis likely that an 11β-reductase inhibitor will be selective for the11β-HSD1 isozyme in vivo. We have found, for instance, thatglycyrrhetinic acid (a known inhibitor of 11β-dehydrogenase) has noeffect on 11β-reductase in vivo [26]. However, we have surprisinglyfound that carbenoxolone, which is known as an inhibitor of the11β-dehydrogenase enzyme, also inhibits 11β-reductase in vivo [26, 27].In preferred embodiments, therefore, the inhibitor is carbenoxolone(3β-(3-carboxyproprionyloxy)-11-oxo-olean-2-en 30-oic acid), or apharmaceutically acceptable salt thereof. The dose of carbenoxolonewhich we used in our studies was 100 mg every 8 hours given orally.

[0014] The invention is hereinafter described in more detail by way ofexample only, with reference to the following experimental proceduresand results and the accompanying figures, in which.

[0015]FIG. 1 is a graph showing dextrose infusion rates (M values) afterplacebo or carbenoxolone administration, in an euglycaemichyperinsulinaemic clamp study carried out on humans;

[0016]FIG. 2 is a graph showing fasting plasma glucose concentration inrats after placebo or carbenoxolone therapy at the daily dosesindicated;

[0017]FIG. 3 is a graph illustrating the effect of estradioladministration to gonadectomised or gonadectomised/adrenalectomised ratson hepatic 11β-HSD1 activity, and mRNA expression for 11β-HSD1 andPEPCK;

[0018]FIG. 4 contains graphs of data obtained from mice which are eitherhomozygous for the wild type 11β-HSD1 allele or which are homozygous fora knockout mutant allele for 11β-HSD1;

[0019]FIG. 5 illustrates 11β-HSD1 enzyme activities and mRNA expressionin undifferentiated and differentiated 3T3-F442A cells;

[0020]FIG. 6 is a graph illustrating the effect of pretreatment withcorticosterone and 11-dehydrocorticosterone with or withoutcarbenoxolone, upon rat hippocampal cell survival on exposure to kainicacid.

EXPERIMENTAL RESULTS IN SUPPORT OF THE INVENTION

[0021] A. Insulin Sensitivity

[0022] A.1 Effect of Carbenoxolone on Insulin Sensitivity in Man

[0023] Consistent with the observations in animal tissues and culturedcells described above, we have established the relative activities of11β-dehydrogenase and 11β-reductase in vivo in different organs in manby measuring the cortisol/cortisone ratio in plasma from selectivevenous catheterisations [28]. In most organs the venous effluentcontains cortisol and cortisone in a ratio of −10:1. However, in renalvein plasma the ratio is 3:1, while in hepatic vein plasma the ratio is55:1, consistent with potent 11β-dehydrogenase activity due to 11β-HSD2expression in the kidney and potent 11β-reductase 11β-HSD1 activity inthe liver. Moreover, cortisone taken orally, and therefore delivered tothe liver via the portal vein, is avidly converted on first pass tocortisol in the peripheral circulation [26, 27]. Using this index of11β-reductase activity we have shown that, in addition to inhibitingrenal 11β-dehydrogenase activity, carbenoxolone (but not glycyrrhetinicacid) [26, 27] inhibits hepatic 11β-reductase activity in vivo in man.

[0024] In the liver, mineralocorticoid receptors are not expressed insignificant numbers, but glucocorticoid receptors are abundant.Surprisingly, the affinity of glucocorticoid receptors for cortisol is10-40 times lower than that of mineralocorticoid receptors [29]. It maybe that, by contrast with the protection of high-affinitymineralocorticoid receptors from cortisol required of 11β-dehydrogenaseactivity in the distal nephron, 11β-reductase activity in the liver isrequired to ensure that low-affinity glucocorticoid receptors getadequate exposure to cortisol. The free circulating concentrations ofcortisol and cortisone are approximately equal, so that a large pool ofcortisone is available for activation in the liver. Such a mechanismwould be analogous to the intra-cellular activation of other members ofthe thyroid-steroid hormone family which circulate in relativelyinactive forms [30], such as testosterone which is converted by5α-reductase to dihydro testosterone, and thyroxine which is convertedby 5′-monodeiodinase to tri-iodothyronine.

[0025] To test this hypothesis, we have used carbenoxolone to inhibithepatic 11β-reductase, and observed changes in hepatic glucocorticoidreceptor activation inferred indirectly from changes in insulinsensitivity. Hepatic insulin sensitivity decreases and hepatic glucoseproduction increases during both pharmacological [31, 32] andphysiological [33] increases in glucocorticoid levels. The results ofthese experiments were published [16] after the filing of our Britishpatent application number 9517622.8 on 29 August 1995.

[0026] Methods

[0027] Seven non-obese (Body Mass Index <25) healthy male Caucasianvolunteers on no medication, aged 27-36 years, were given carbenoxolone(100 mg every 8 hours orally) or matched placebo for seven days in arandomised double-blind cross-over design, with phases separated by atleast 4 weeks. On the seventh day of each phase euglycaemichyperinsulinaemic clamp studies with measurement of forearm glucoseuptake were performed, as described in detail in reference 16, thecontent of which is incorporated herein by reference.

[0028] Results

[0029] Carbenoxolone administration was associated with no change infasting glucose concentration but a fall in fasting insulinconcentration. During the euglycaemic clamp, carbenoxolone enhanced themetabolic clearance rate of glucose (M values 41.1±2.4 μb 5 mol/kg/minfor placebo versus 44.6±2.3 for carbenoxolone, p<0.03) (FIG. 1). Nodifference was detected in peripheral glucose uptake as measured byforearm glucose uptake, but this measurement was less precise due toinsulin-induced vasodilatation [34] and the mental stress induced by therestraint of the study conditions.

[0030] Discussion

[0031] These data show that carbenoxolone increases whole-body insulinsensitivity. This could have resulted from either increased suppressionof hepatic glucoase production as a reflection of increased hapaticinsulin sensitivity, or increased peripheral utilisation of glucose as areflection of increased peripheral insulin sensitivity. The absence of achange in forearm glucose uptake with carbenoxolone suggests that theformer mechanism predominates. However, the imprecision of thismeasurement raises the possibility that there is a contribution fromenhanced peripheral insulin sensitivity which we have not detected. Amore detailed discussion of these data has now been published [16].

[0032] It is unlikely that carbenoxolone affects insulin sensitivity bya mechanism independent of its effect on 11β-reductase. Carbenoxoloneinhibits other enzymes which metabolise cortisol, notably 5β-reductase[35], but this effect would increase intra-hepatic cortisolconcentrations and reduce insulin sensitivity. In the absence ofcorticosteroids, carbenoxolone at this dose has no documented effects invivo and low affinity for corticosteroids receptors in vitro [36], sothat a direct action of carbenoxolone to increase insulin sensitivity isunlikely. Indeed, previous experiments suggest that, at higherconcentrations (mmol/l), carbenoxolone without corticosteroidantagonises the action of insulin in adipocytes [37]. Blood pressure andforearm blood flow were not elevated by carbenoxolone in this study, andeven if they were this might be associated with reduced insulinsensitivity [34]. Finally, other neurohumoral changes consequent on therenal effects of carbenoxolone (suppressed plasma renin, aldosterone,and potassium concentrations) are not known to influence insulinsensitivity directly, and in the context of oral sodium loading wereassociated with decreased insulin sensitivity [38].

[0033] From these observations we infer that basal 11β-reductaseactivity plays a role in maintaining adequate exposure of glucocorticoidreceptors to cortisol in human liver. The circulating pool of cortisoneis therefore physiologically important as a source of activeglucocorticoid in sites where 11β-reductase is expressed.

[0034] A.2 Effect of Carbenoxolone on Insulin Sensitivity in the Rat

[0035] Following our studies of the effects of carbenoxolone in healthyhumans, we have performed unpublished studies of the effects ofcarbenoxolone in intact healthy rats. Consistent with the observationsin human, we have shown that rat hepatocytes in primary culture express11β-HSD1 which has predominant 11β-reductase activity [15]. We have alsoshown that the circulating concentration of 11-dehydrocorticosterone(measured by gas chromatography and mass spectrometry) is 40-50 nmol/l(Dr. R. Best, unpublished observation) so that there is a substantialpool of inactive 11-ketosteroid available for activation tocorticosterone in sites where 11β-reductase is active.

[0036] Methods

[0037] Intact male Wistar Hans rats of body weight 200-250 g weretreated with daily subcutaneous injection of 1 ml of either 0.9% saline(vehicle) or carbenoxolone (made up to 1.3 or 10 mg/ml in 0.9% saline)for 14 days. They were fasted from 4 pm and blood obtained by tail-nickat 9 am the next day. Plasma glucose was measured by a glucose oxidasemethod on a Beckman CX-3 autoanalyser.

[0038] Results

[0039] Carbenoxolone produced a dose-dependent fall in tasting glucoseconcentrations (FIG. 2).

[0040] Discussion

[0041] These experiments are preliminary, since it is now our intentionto assess insulin sensitivity and glucose tolerance in rats treated withcarbenoxolone. However, the fall in fasting plasma glucose is consistentwith inhibition of hepatic gluconeogenesis with or without enhancedperipheral glucose uptake in rats treated with the 11β-reductaseinhibitor carbenoxolone.

[0042] A.3 Effect of Estradiol-mediated Inhibition of Hepatic 11β-HSD1Expression on Insulin Sensitivity in the Rat

[0043] Having established the effects of a competitive pharmacologicalinhibitor of 11β-reductase activity on insulin sensitivity and glucoselevels in man and rat, the object of this study was to examine theeffects of down-regulation of transcription and translation of the11β-HSD1 enzyme. The results of these studies have been presented atscientific meetings during 1996 but are not yet published.

[0044] 11β-HSD1 expression is regulated by a variety of hormones in vivo[26]. In the rat liver, 11β-HSD1 shows pronounced sexual dimorphism,with lower activity in females. Indeed, chronic estradiol administrationalmost completely suppresses hepatic 11β-HSD1 expression in both maleand female rats [39-41]. This regulation appears to be tissue- andisozyme-specific, as estrogen does not attenuate 11β-HSD1 expression inthe hippocampus or 11β-HSD2 activity in the kidney [41]. The aim of thisstudy was therefore to examine the contribution which reactivation ofglucocorticoids by hepatic 11β-HSD1 makes to the expression ofliver-specific glucocorticoid modulated genes in the rat by exploitingthe selective suppression of hepatic 11β-HSD1 by estradiol in vivo.

[0045] Methods

[0046] In-vivo Studies

[0047] Male Han Wistar rats (200-250 g) underwent gonadectomy and eitherbilateral adrenalectomy of sham-operation under halothane anaesthesia.For estradiol administration, silicone elastomer capsules (1.95 mminternal diameter, 3.125 mm external diameter). (Dow CorningCorporation, Midland, Mich., U.S.A.) containing 17β-estradiol (Sigma,Poole, UK) were implanted subcutaneously. For animals treated for 42days with estradiol, the capsules were removed and replaced after 21days. Control animals were implanted with blank capsules.Adrenalectomised rats were maintained on 0.9% saline. Rats were killed10, 21 or 42 days after surgery. Liver and hippocampus were removed anddissected on ice for assay of 11β-HSD activity and an aliquot of liverwas frozen on dry ice and stored at −80° C. until extraction of RNA.

[0048] Quantitation of 11β-HSD Activity in In Vivo Experiments

[0049] Tissues were homogenised in Krebs-Ringer bicarbonate buffer with0.2% glucose, pH 7.4, and assayed as described previously [40].Homogenates (6.25 μg of liver protein 250 μg of hippocampal protein)were incubated with 200 μM NADP (Sigma, Poole, UK) and 12 nM[³]-corticosterone (specific activity 84 Ci/mmol; AmershamInternational, Aylesbury, UK) in a total volume of 250 μl withKrebs-Ringer buffer supplemented with 0.2% bovine serum albumin for 10min at 37° C. 11β-dehydrogenase activity was quantified in this assay asa measure of active enzyme since 11β-reductase is unstable inhomogenates. Steroids were extracted with ethyl acetate and separated byHPLC with on-line β counter. 11β-HSD activity was expressed asconversion of corticosterone to 11-dehydrocorticosterone, aftercorrection for apparent conversion in incubates without enzyme (<3%)

[0050] Extraction and Analysis of mRNA

[0051] Total RNA was extracted from liver tissue by the guanidiniumthiocyanate method, as described [40] and resuspended indiethylpyrocarbonate-treated water. RNA concentration and purity wasassayed spectrophotometrically 20 μg aliquots were separated on a 1.2%agarose gel containing 2% formaldehyde. RNA was blotted ontonitrocellulose membranes (Hybond-N, Amersham International, UK),prehybridized in 6 ml phosphate buffer (0.2 M NaH₂PO₄, 0.6 M NaH₂PO₄, 5mM EDTA). 3 ml 20% SDS and 100 μg denatured herring testis DNA (Sigma,Poole, UK) for 2 h at 55° C. and hybridized at 55° C. overnight in thesame solution containing rat 11β-HSD1 or PEPCK cDNA, labelled with³²P-dCTP using a random primed DNA labelling kit (Boehringer Mannheim UKLtd., Lewes, UK). Three 20 min washes were carried out at roomtemperature in 1×SSC (0.3 M NaCl, 0.03 M sodium citrate), 0.1% SDSfollowed by a stringent wash at 55° C. for 30 min in 0.3×SSC, 0.1% SDS.Filters were exposed to autoradiographic film for 1-4 days (adjusted toensure the signal density was within the linear range). Filters wererehybridised with similarly labelled 7S cDNA or transferrin RNA cDNAprobes to control for loading, as previously described [40] (transferrinmRNA expression is not altered by estradiol or glucocorticoid). Opticaldensity was determined using a computer-driven image analysis system(Seescan plc, Camps, UK). Values were expressed as a percentage ofcontrol levels.

[0052] Statistics

[0053] Data are the means±SEM of 5-10 replicates (indicated in figurelegends). Data were compared by ANOVA and Newman-Keuls post-hoc test orby Student's unpaired t-test, as appropriate. Statistical tests werecarried out on absolute data, but the figures display data as percent ofvalues for control animals to improve clarity.

[0054] Results

[0055] Effect of Chronic Estradiol Treatment on 11β-HSD1 Activity andmRNA Expression

[0056] Estradiol administration for 10, 21 and 42 days to gonadectomisedmale rats resulted in marked decreases in hepatic 11β-HSD activity (FIG.3). However, 11β-HSD activity was not completely abolished sinceprolonged (60 min) incubation of liver homogenates from rats givenestradiol for 42 days showed detectable (12.2±14.7%) conversion ofcorticosterone to 11-dehydrocorticosterone 11β-HSD1 mRNA expression fellto undetectable levels after 21 and 42 days of estradiol treatment.11β-HSD activity in the hippocampus was not altered by estradioltreatment at 21 or 42 days (data not shown).

[0057] Effect of Estradiol on Hepatic Glucocorticoid-inducible GeneExpression

[0058] To examine the effect of attenuated hepatic 11β-HSD1 activity onlocal glucocorticoid action, the expression of liver-specificglucocorticoid-inducible genes was measured after 42 days of estradiol.PEPCK mRNA expression was significantly reduced by estradiol treatment(FIG. 3).

[0059] To examine whether these effects of estradiol on hepatic geneexpression were mediated directly or via alterations in glucocorticoidaction in the liver, the effects of estradiol in adrenalectomisedanimals or sham-operated controls was examined. Animals were killedafter 21 days. Plasma corticosterone values confirmed the adequacy ofadrenalectomy (data not shown). Adrenalectomy increased hepatic 11β-HSD1gene expression and activity compared with sham-operated controls (FIG.3). Estradiol reduced liver 11β-HSD1 mRNA and activity inadrenalectomised rats, and although mRNA expression was higher thanestradiol-treated adrenally-intact rats, it remained significantlyreduced compared with untreated controls.

[0060] Hepatic PEPCK gene expression was decreased after 21 days ofestradiol treatment, albeit to a slightly lesser extent than after 42days (FIG. 3). Unsurprisingly, adrenalectomy also attenuated hepaticPEPCK gene expression, but in adrenalectomised rats estradiol no longerreduced, but indeed increased PEPCK mRNA levels when compared withadrenalectomy alone.

[0061] Discussion

[0062] Estradiol administration for both 21 and 42 days producedundetectable hepatic 11β-HSD1 mRNA expression and markedly reduced11β-HSD activity (although this remained clearly measurable), confirmingprevious studies [41]. The discrepancy between mRNA expression andactivity might be due to much slower turnover of 11β-HSD1 protein or,more likely, to the transcription and translation of residual low levelsof 11β-HSD1 mRNA, undetectable by northern analysis. This effect of sexsteroids is relatively specific to the liver, since hippocampal 11β-HSD1was unaffected, and previous studies have shown that 11β-HSD2 is notattenuated by estrogens [22]. Thus the advantage of exploiting estrogendown-regulation of hepatic 11β-HSD1, as opposed to conventionalcarbenoxolone or other inhibitors of the enzyme, is that 11β-HSD2 isunaffected.

[0063] Following 42 days of estradiol treatment, hepatic expression ofmRNA encoding PEPCK was clearly reduced. In principle, this effect ofchronic estrogen treatment may be explained in several ways: (i)estrogen may act directly to repress hepatic gene expression; (ii)estradiol may alter corticosteroid metabolism in the liver. Reducedhepatic 11β-HSD1-mediated reactivation of otherwise inert11-dehydrocorticosterone is anticipated to reduce expression of theseglucocorticoid-sensitive transcripts; (iii) there may be other indirecteffects of chronic estradiol administration.

[0064] For PEPCK direct inhibition by estradiol appears unlikely, sincethis transcript was induced by estrogen in adrenalectomised rats. Theloss of the attenuating effect of estradiol on PEPCK expression inadrenalectomised rats suggests a requirement for glucocorticoids in theprocess, rather than any other indirect effect. Glucocorticoids increaseexpression of PEPCK in the liver [17] and so this pattern of changes iscompatible with the glucocorticoid control of expression of this gene.The effect of estradiol in intact rats was as potent as adrenalectomyalone in attenuating PEPCK mRNA expression. These data are thereforeconsistent with the effects of estradiol being indirectly mediated viathe marked loss of 11β-HSD1 11β-reductase activity, which thusattenuates intrahepatic glucocorticoid regeneration. Thus the11β-reductase activity of 11β-HSD1 is likely to be of key importance inproducing sufficiently high intrahepatic corticosterone levels toelevate PEPCK above the minimum levels maintained by basal(non-glucocorticoid) factors.

[0065] A.4 Effect of Transgenic Knockout of 11β-HSD1 Expression

[0066] Although the effects of carbenoxolone and estrogen-induceddownregulation of 11β-HSD1 expression provide strong support for ourhypothesis that 11β-reductase inhibition will enhance insulinsensitivity and reduce hepatic glucose production, both of these agentsare potentially confounded by other actions, including direct effects on11β-HSD2 (carbenoxolone) and other effects on the liver (estrogen). Themost clear-cut evidence of the importance of a specific protein can nowbe tested most effectively using transgenic technology. We have nowproduced a transgenic mouse which is homozygous for a mutant allelecarrying a targetted disruption of the 11β-HSD1 gene. Some of theseresults have been presented at scientific meetings during 1996 but havenot yet been published.

[0067] Methods

[0068] A mouse genomic library constructed from isogenic OLA129 DNA inthe lambda vector GEM12 was screened with a rat 11β-HSD1 cDNA probe. Alambda phage containing a 14 kb insert encompassing exons 2 to 5 of themouse 11β-HSD1 gene was cloned and subjected to extensive restrictionmapping and sequence analysis. Using this information, a replacementvector 16 KpnβA was constructed using the pBS-KpnA cassette. The vectorconsisted of the neomycin resistance gene under the control of the humanβ-actin promoter and followed by the SV40 polyadenylation signal. The6.5 kb long 3′ homology arm was subcloned from intron D. The 1.2 kbshort homology arm was synthesised by PCR using high fidelity Ultma Taqpolymerase, 129 mouse genomic DNA and primers bearing nested Sac I andSpe I restriction sites and located in exon 1 and introl B. An externalprobe was synthesised by PCR. Using this probe, a specific recombinationevent generates a 1.5 kb BamHI fragment instead of a 3 kb fragmentexpected for the wild type gene.

[0069] Following transfection of the 16 kpnβ A replacement constructinto ‘CGR 8’ embryonic stem cells, of 368 neomycin-resistant coloniesscreened, only one showed homologous recombination at the 5′ end. Thespecificity of the recombination event was tested by Southern blothybridisation after BamHI restriction. Specificity of restriction at the3′ end was also confirmed by hybridisation with an internal probe afterthree different restrictions.

[0070] Positive emoryonic stem cells were injected into C57B1/6blastocysts to generate onimeric offspring who were crossbred toestablish a colony of homozygotes for the knockout allele.

[0071] Results

[0072] Homozygotes for the mutant allele are morphologically intact andfully fertile. No survival bias is conferred by the mutant allele.However, neither 11β-HSD1 mRNA nor enzyme activity (techniques as above)were detected in mutant homozygotes, and reduced mRNA and activity weredetected in heterozygotes. Plasma corticosterone levels are not alteredin mutants, but the ratio of corticosterone to 11-dehydrocorticosteronein urine is markedly reduced. Homozygous mutant mice are also unable toconvert 11-dehydrocorticosterone to corticosterone in vivo and are thusresistant to the thymic involution seen with 11-dehydrocorticosterone inwild-type control mice.

[0073] Animals were fasted for 48 hours before sacrifice and collectionof trunk blood. Plasma glucose was measured by a glucose oxidase method(see above). Fasting plasma glucose concentrations were lower in mutantanimals (FIG. 4).

[0074] Measurement of enzymes responsible for hepaticglycolysis/gluconeogenesis revealed no difference between wild type andmutant animals in the fed state. However, on fasting the mutant animalsfailed to show the normal induction of glucose-6-phosphatase and PEPCK(FIG. 4). Glucokinase was not altered.

[0075] Discussion

[0076] These data demonstrate the importance of 11β-HSD1 in hepaticglucose metabolism and insulin sensitivity. Glucose-6-phosphatase andPEPCK are two gluconeogenic enzymes which are down-regulated by insulinand up-regulated by glucocorticoid exposure [1]. In the presence ofinsulin (in fed animals) there is no change in expression of theseenzymes, confirming that insulin regulation is dominant. However, instarved animals with low insulin levels there is a failure of inductionof these enzymes consistent with a failure of corticosterone-dependentinduction. In animals in which the mutant vector has recombinedspecifically to knockout only 11β-HSD1, and in which plasmacorticosterone is maintained by normal hypothalamic-pituitary-adrenalfeedback these changes can be attributed to impaired intra-hepaticconversion of 11-dehydrocorticosterone to corticosterone.

[0077] A.5 Role of 11β-HSD1 in Adipocyte Maturation

[0078] Glucocorticoids are involved in triggering adipocytedifferentiation from uncommitted adipoblasts into committedpreadipocytes. Glucocorticoid excess in humans causes profound changesin both adipose tissue distribution and metabolism [18]. In Cushing'ssyndrome, omental fat shows adipose cell hyperplasia, associated withenhanced lipoprotein lipase and glycero-phosphate dehydrogenase activityin homogenised adipose tissue cortisol and corticosterone are oxidisedto cortisone and 11-dehydrocorticosterone, indicating the presence of11β-HSD [42] which might modify glucocorticoid action in a sites- anddevelopmentally-specific manner. We have (i) examined the expression of11β-HSD enzyme activity and 11β-HS1 and mRNA in isolated primary ratadipocytes; and (ii) determined whether 11β-HSD1 is expressed in afibroblast derived clonal cell line. 3T3-F442A which can be induced intomature adipocytes in presence of fetal calf serum and insulin. Theresults of this work have not been published.

[0079] Methods

[0080] Animals

[0081] Epididymal adipose tissue was excised from adult male Wistarrats. Adipose tissue was washed several times in Phosphate BufferedSaline, trimmed of large blood vessels and minced. Tissue was incubatedin Krebs-Ringer buffer (KRB) containing collagenase II (2 mg/ml) (Sigma,UK) at 37° C. for 40 min, and the digested material passed through a 250μm nylon filter and briefly centrifuged. The separated adipocyte(floating) and stromal vascular (pellet) fractions were collected for(i) RNA extraction and northern blot as described above and (ii)11β-dehydrogenase bioactivity assay as described above, using finalconcentrations of 500 μg protein/ml. 200 μM cofactor (NADP or NAD), and12 nM 1, 2, 6, 7- ³H-corticosterone in a total volume of 250 μlKrebs-Ringer buffer for 15 min at 37° C.

[0082] Cell Culture

[0083] Clonal preadipocyte cell lines 3T3-F442A, (kindly provided by Dr.Pairault Henri Modor Hospital, Creteil, France) were plated at a densityof 10⁴ cells/100 mm diameter dishes in ‘basal medium’ (Dulbecco'smodified Eagle's medium (DMEM), supplemented with 10% newborn calfserum, penicillin 200 U/ml and streptomycin 50 μg/ml). To differentiateconfluent 3T3-F442A cells, basal medium was replaced with‘differentiation medium’ (DMEM, 10% fetal calf serum and insulin, 5μg/ml) and maintained in this for 11 days with the medium changed every48 h.

[0084] 11β-Dehydrogenase and 11β-reductase activities were measured inintact cells by addition of 25 nM corticosterone with labelled³H-corticosterone or 25 nM 11-dehydrocorticosterone with labelled³H-11-dehydrocorticosterone. Intraconversion of corticosterone and11-dehydrocorticosterone was assessed as above at 3, 8 and 24 h afterthe addition of steroids.

[0085] Results

[0086] Extracts from homogenized rat adipocytes showed 11β-HSD activity,as demonstrated by conversion of corticosterone to11-dehydrocorticosterone. 11β-HSD1 was transcribed in whole whiteadipose tissue and isolated adipocytes as a single, approximately 1.6 kbspecies of mRNA, of similar size to the hepatic species, thoughexpression was lower than in liver 11β-HSD1 transcripts were alsoexpressed in the stromal vascular fraction (data not shown).

[0087] 3T3-F442A cells were induced to differentiate and after 10days, >90% cells were differentiated as determined by visible lipidaccumulation. Associated with this differentiation there was a markedincrease in 11β-HSD1 mRNA expression and 11β-reductase activity whichoccurred late (8 days after addition of differentiation medium) and inassociation with induction of GPDH expression (FIG. 5).

[0088] Discussion

[0089] In this study, we have demonstrated 11β-HSD1 gene expression inrat adipose tissue and isolated rat adipocytes, as well as the stromalvascular preadipocyte fraction, in agreement with data demonstrating11β-HSD activity in the adipose component of the mammary gland [42]. Bycontrast with data obtained in homogenised tissue and in keeping withfindings in other whole cell preparations [15, 22], we have found inwhole cells that 11β-HSD1 operates as an 11β-reductase rather than11β-dehydrogenase enzyme in addition, in the 3T3-F442A cell line, wehave demonstrated that 11β-HSD1 expression is regulated in adifferentiation-dependent manner. 11β-HSD1 thus has the characteristicsof a ‘late’ differentiation gene. Moreover, the regulated expression of11β-HSD1 paralleled that of GPDH, a glucocorticoid-sensitive late markerof adipocyte differentiation.

[0090] These results support the hypothesis that 11β-reductase plays aphysiological role in converting 11-keto steroids to 11β-hydroxysteroids and thus amplifying glucocorticoid activity. In the adipocytethis may explain the induction of GPDH. Although inhibition of11β-reductase would not be expected to influence the earlydifferentiation of adipocytes, it may influence the biochemicalphenotype of differentiated cells, specifically in relation to lipidmetabolism.

[0091] B Neuronal Effects of 11β-Reductase Inhibitors

[0092] Several studies have demonstrated 11β-HSD activity,immunoreactivity and mRNA expression in hippocampal neurons [43]45.Administration of 11β-HSD inhibitors alters functional activity in thehippocampus in vivo [46], although the mechanisms underpinning thiseffect are obscure.

[0093] Previous studies have shown that in homogenates of hippocampus,both dehydrogenation and reduction occur [44]. but the reactiondirection in intact cells was previously unknown. We have now examined11β-HSD activity and its function in primary cultures of fetalhippocampus cells. The results of these experiments were published [22]after the filling of our patent application No. 9517622.8 on 29 Aug.1995. Further details of the experiments will be found in reference 22,the contents of which are incorporated herein by way of reference.

[0094] Methods and Results

[0095] Hippocampi were dissected from Wistar rat embryos on day 18.5 ofpregnancy and cultured in DMEM with supplements, as described [22].These cells expressed 11β-HSD1 mRNA, and homogenised cells performedeither 11β-dehydrogenase or 11β-reductase activity. However, in wholecells in culture, only 11β-reductase activity was demonstrated. This11β-reductase activity could be almost abolished by the addition ofcarbenoxolone (10⁻⁶ M) [22].

[0096] To assess the influence of 11β-reductase activity onneurotoxicity, cells were exposed to kainic acid and cell survivaldetermined. Both corticosterone and 11-dehydrocorticosterone potentiatedkainic acid neurotoxicity. However, carbenoxolone did not alter theeffect of corticosterone, but protected the cells from the potentiatingeffect of 11-dehydrocorticosterone (FIG. 6).

[0097] Discussion

[0098] These data demonstrate that 11β-HSD1 is an 11β-reductase enzymein rat hippocampus and that the conversion of 11-ketosteroids to11-hydroxysteroids potentiates the neurotoxic action of glucocorticoids.Importantly, they show that inhibition of 11β-reductase activity canprotect the cells from the damage that occurs due to reactivation of11-dehydrocorticosterone. This supports the hypothesis that inhibitionof 11β-reductase in human brain would prevent reactivation of cortisoneinto cortisol and protect against deleterious glucocorticoid-mediatedeffects on neuronal survival and other aspects of neuronal function,including cognitive impairment, depression, and increased appetite.

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1. The use of an inhibitor of 11β-reductase in the manufacture of amedicament for the control of 11-keto steroid conversion to11β-hydroxysteroid in vivo.
 2. The use according to claim 1, for thecontrol of cortisone conversion into cortisol in humans.
 3. The useaccording to claim 2, for lowering hepatic cortisol concentration. 4.The use according to claim 3, for inhibiting hepatic gluconeogenesis. 5.The use according to claim 2, for lowering intracellular cortisolconcentration.
 6. The use according to claim 5, for increasing insulinsensitivity in adipose tissue.
 7. The use according to claim 5, forincreasing insulin sensitivity in muscle.
 8. The use according to claim5, for the prevention or reduction of neuronal dysfunction orloss/cognitive impairment due to glucocorticoid potentiatedneurotoxicity or neural dysfunction or damage.
 9. The use of aninhibitor of 11β-reductase in the manufacture of a medicament forproducing multiple therapeutic effects in a patient to whom themedicament is administered, said therapeutic effects including aninhibition of hepatic gluconeogenesis, an increase in insulinsensitivity in adipose tissue and muscle, and the prevention of or areduction in neuronal dysfunction, damage or loss due to glucocorticoidpotentiated neurotoxicity.
 10. The use according to any preceding claim,for the treatment of diabetes mellitus, impaired glucose tolerance, orglucocorticoid associated cognitive or affective disorder.
 11. The useaccording to any preceding claim, in which the 11β-reductase inhibitoris carbenoxyolone (3β-(3-carboxypropionyloxy)-11-oxo-olean-2-en 30-oicacid), or a pharmaceutically acceptable salt thereof.
 12. A method oftreatment of a human or animal patient suffering from a conditionselected from the group consisting of hepatic insulin resistance,adipose tissue insulin resistance, muscle tissue insulin resistance,neuronal loss due to glucocorticoid potentiated neurotoxicity, and anycombination of the aforementioned conditions, the method comprising thestep of administering to said patient a medicament comprising apharmaceutically active amount of an inhibitor of 11β-reductase.
 13. Amethod according to claim 12, wherein said inhibitor is selected fromthe group consisting of carbenoxolone(3β-(3-carboxypropionyloxy)-11-oxo-olean-2-en 30-oic acid), andpharmaceutically acceptable salts of carbenoxolone.